Aqueous solutions containing two different polymers can be immiscible when mixed together above critical concentrations. The resulting interface of aqueous two-phase systems (ATPS) exhibits a remarkably low surface tension (˜1-100 μN m−1), allowing for biomolecules to readily traverse between the phases without denaturation [Ryden et al. (1971) J. Colloid Interface Sci. 37:219-222; Liu et al. (2012) Langmuir 28:3831-3839]. For this reason, ATPS are of great interest for the partitioning and self-assembly of biomaterials by exploiting their affinity for one of the polymer phases [Alberts son (1986) “Partitioning of Cell Particles and Macromolecules” Wiley, N.Y.; Walter et al. (1991) Anal. Biochem. 197:1-18; Azevedo et al. (2009) Trends Biotechnol. 27:240-247]. Over the past decade various microfluidic devices have used polymer solutions such as poly(ethylene glycol) (PEG) and dextran to generate aqueous two-phase flows capable of partitioning cells [Yamada et al. (2004) Biotechnol. Bioeng. 88:489-494; Nam et al. (2005) Biomed. Microdevices 7:189-195; Tsukamoto et al. (2009) Analyst 134:1994-1998; SooHoo et al. (2009) Biomed. Microdevices 11:323-329; Frampton et al. (2011) Biomed. Microdevices 13:1043-1051], proteins [Munchow et al. (2007) Lab Chip 7:98-102; Meagher et al. (2008) Lab Chip 8:527-532], and DNA [Hahn et al. (2011) Soft Matter 7:6320-6326].
Recently there has been much interest in containing ATPS within droplets to mimic the differences in local composition within the cytoplasm of cells, known as microcompartmentation [Munchow 2007]. This interest has been motivated by evidence that aqueous phase-separation is an important mechanism for cellular microcompartmentation, [Walter et al. (1995) FEBS Lett. 361:135-139; Ge et al. (2009) J. Am. Chem. Soc. 131:9094-9099], including the observation that P granules in germ cells are liquid droplets, and therefore some intracellular compartments are entirely aqueous in composition [Brangwynne et al. (2009) Science 324:1729-1732]. An acoustically levitated aqueous two-phase droplet was used to demonstrate the affinity partitioning of biotinylated liposomes [Santesson et al. (2004) Anal. Chem. 76:303-308]; however the millimetric droplet size is incommensurate with cellular length scales. Synthetic cells have been developed by encapsulating ATPS inside of spherical lipid bilayer membranes. [Helfrich, et al. (2002) J. Am. Chem. Soc. 124:13374-13375] In these systems, proteins could be preferentially sorted into the PEG or dextran-rich phase via affinity partitioning [Long et al. (2005) Proc. Natl. Acad. Sci. U.S.A. 102:5920-5925] or by tuning the pH. [Dominak et al. (2010) Langmuir 26:5697-5705]. However, this encapsulation method, while effective, results in a wide variety of vesicle sizes and morphologies, such as multilamellar vesicles and vesicles where each phase is additionally encapsulated by separate lipid bilayers [Long 2005]. The concentrations of the encapsulated polymers and molecules also tend to vary between vesicles [Dominak et al. (2007) Langmuir 23:7148-7154], and during osmotic deflation the lipid bilayer is wetted by both phases [Li et al. (2008) J. Am. Chem. Soc. 130:12252-12253] and becomes unstable, resulting in vesicle budding [Long et al. (2008) J. Am. Chem. Soc. 130:756-762; Li et al. (2011) Proc. Natl. Acad. Sci. U.S.A. 108:4731-4736; Li et al. (2012) Phys. Chem. B 116:1819-1823] and fission [Andes-Koback et al. (2011) J. Am. Chem. Soc. 133:9545-9555].
Formation of ATPS inside of vesicles has not yet been demonstrated in a microfluidic device, although a recent work showed successful encapsulation of a single phase of dextran inside monodisperse lipid membranes within a micro-channel [Matosevic et al. (2011) J. Am. Chem. Soc 133:2798-2800]. Without resorting to lipid membranes [Matosevic (2011)] or levitation [Santesson 2004], a double emulsion is required to contain an ATPS within a microdroplet. Obtaining even a single emulsion (e.g., a dextran drop inside a continuous PEG phase) within a microfluidic ATPS is not trivial, however, as the low surface tension makes it difficult to break up streams or jets into individual microdroplets. Currently, single emulsion (w/w) microfluidic ATPS have been achieved by electrically [Song et al. (2007) J. Chromatogr., A, 1162:180-186; and Ziemecka et al. (2011a) Lab Chip 11:620-624] or mechanically [Shum et al. (2012) Biomicrofluidics 6:012808; Sauret et al. (2012) Appl. Phys. Lett. 100:154106] perturbing a jet, or by fabricating rounded multi-leveled microchannels to induce flow instabilities [Lai et al. (2011) Lab Chip 11:3551-3554].
Very recently, all-aqueous (w/w/w) double emulsions were obtained in a microchannel using electrical [Ziemecka (2011b) Soft Matter 7:9878-9880] and mechanical pulsations [Sauret (2012); Song et al. (2012) Langmuir 28:12054-12059]. Interestingly, both PEG-in-dextran-in-PEG and dextran-in-PEG-in-dextran systems were possible, although these types of all-aqueous double emulsions are inherently unstable and break down to their phase separated states within seconds [Lai (2011)] or minutes [Ziemecka (2011b)]. A dextran-in-PEG-in-oil (w/w/o) ATPS, on the other hand, is both stable and conducive to droplet pinch-off due to the large surface tension of the water/oil interface. Previously, aqueous two-phase droplets in an oil microchannel have been obtained using a T-junction [Vijayakumar et al. (2010) Chem. Sci. 1:447-452; Lee et al. (2012) Biomicrofluidics, 6, 022005] or flow-focusing cross junctions [Ma et al. (2012) Small, 8:2356-2360].
The present invention makes possible generation of femtoliter-volume, aqueous two-phase droplets in an oil microchannel. In contrast to other reports of aqueous two-phase microdroplets, which rely on continuous flows/jets and high-frequency droplet formation, [Sauret (2012); Ziemecka (2011b); Song (2012); Vijayakumar (2010); Lee (2012); Ma (2012)] the devices herein and methods of the invention exploit the interfacial tension between the oil and aqueous phases to generate ultrasmall two-phase droplets with a well-defined time zero and without crossflow in the oil phase. This allows individual droplets to be monitored for extended times and to carry out programmed sequential phase transitions. Due to the large surface area to volume ratio of the microdroplets, single-phase droplets with initially low polymer concentrations transition to two-phase droplets during evaporation, and subsequently transition further to core-shell microbeads. These phase transitions are fully reversible by rehydration via fusion with an additional droplet of pure water. The controlled generation and interconversion of single-phase and two-phase microdroplets allows dynamic microcompartmentation and affinity partitioning, and the reversible creation of core-shell microbeads allows controlled delivery of encapsulated biomaterials.